This invention relates to certain compounds containing a trioxane moiety which have cytotoxic and antitumour activity and their use in the treatment of cancer, processes for preparing such compounds and pharmaceutical compositions containing such compounds.
The compound artemisinin, also known as ginghaosu (1), is a tetracyclic 1,2,4-trioxane occurring in Artemisia annua. Artemisinin and its derivatives dihydroartemisinin (2), artemether (3) and sodium artesunate (4) are routinely used for the treatment of malaria and are particularly effective against cerebral malaria. 
Different modes of action have been proposed by various groups to account for the action of artemisinin and its derivatives in treating malaria (Posner et al., J.Am.Chem.Soc.1996,118,3537; Posner at al., J.Am.Chem.Soc.1995,117,5885; Posner at al., J. Med.Chem.1995,38,2273). Whilst the mode of action of artemisinin as an antimalarial has not been unequivocally established, it has been demonstrated that the peroxide linkage is essential for expression of activity. One proposal embodies cleavage of the endoperoxide bridge by intraparasitic heme iron (II) to generate unstable free radical intermediates which alkylate malaria proteins (see Scheme 1 below). 
Recently, the isolation of a covalent adduct formed between artemisinin and a heme model MnIITPP tends to support this proposal (Robert et al, J.Am.Chem.Soc. 1997,119,5968). Formation of artemisinin-heme and artemisinin-protein adducts have also been reported when P. falciparum infected erythrocytes have been incubated in vitro with radiolabelled artemisinin (Hong et. al., Mol.Biochem.Parasitol.,1994,63,121; Asawamahasakda et al., Antimicrob.Agents Chemother., 1994,38,1854). However, attention has also focused on perferryl iron as the active species as this has been unambiguously detected when artemisinin analogues have been incubated with iron(II). Its postulated mode of formation is rather curious in that it is presumed to arise via a reductive scission of the peroxide bond by iron(II) (see Scheme I).
Based on a careful analysis of products obtained from artemisinin on treatment both with iron (II) and iron (III), it has been shown that the trioxane unit acts as a source of free hydroperoxide or equivalent, which is then capable of generating hydroxyl or alkoxy radicals, or of perferryl iron by direct coordination to the iron(II) site in ferroheme according to well-established models (Haynes et al., Today""s Life Science,1993,14; Tetrahedron Lett.1996,37,253;1996,37, 257). These are set out in Scheme 2 below. 
Peroxides in general are biologically active. Tert-butylhydroperoxide is used as a potent inhibitor of bacterial growth on fish, although it is quite toxic towards many living organisms. The root growth inhibitor of Formula A below also has a peroxide bridge in the ring and the inhibitory action on the plant root is correlated with the peroxide bridge. Recently, the simple cyclic peroxide of Formula B below has been studied as a candidate for radical releasing drugs, because these compounds are known to generate hydroxyl radicals by heat stimuli. 
Certain artemisinin derivatives which contain a peroxide moiety have also been tested for biological activity other than antimalarial activity. For instance, the cytotoxicity of artemisinin, dehydroartemisinin, artemisitene, arteether, ethylperoxyartemisitene and an ether dimer of artemisinin to Ehrlich ascites tumor cells has been reported (Beekman et al., Phytother.Res.,1996,10,140; Woerdenberg et al., J.Nat.Prod.,1993,56,849). Selective cancer cell cytotoxicity from exposure to dehydroartemisinin and holotransferrin, a non-heme iron-transport protein saturated with iron, has also been disclosed (Lai et al.,Cancer Lett.,1995,91,41 and U.S. Pat. No. 5,578,637) with the drug combination being approximately 100 times more effective on molt-4 cells than lymphocytes.
It is known that some biologically active molecules contain chemical groups which enable the molecule to bind to DNA. The method by which DNA-binding occurs will depend upon the overall structure of the molecule and the nature of the chemical groups contained within the molecule.
For instance, the major and minor grooves of double helical DNA are occupied by water under physiological conditions. However, certain oligopeptidic compounds, such as netropsin and distamycin can displace water molecules and form strong hydrogen bonds with hydrophilic groups along the DNA strands. The crescent-shaped structures of netropsin and distamycin can make them fit tightly into the helical structure of DNA.
Alternatively, some compounds contain groups which are capable of intercalating with DNA. Intercalators are flat aromatic compounds which insert between the bases of DNA, the ensemble being held together by hydrophilic and xcfx80xe2x80x94xcfx80 interactions. Well characterised examples of intercalators are provided by anthracyclines, such as adriamycin and daunomycin, which are used for treatment of cancer, and acridines, such as amascrine, which is used for treating acute leukaemia and malignant lymphomas, the antitumour activity is associated with the intercalating property of these compounds.
The technique of incorporating minor groove binding agents related to netropsin or distamycin, or intercalating agents to free-radical generators or electrophilic alkylating agents as a means of inducing DNA strand cleavage is well known. For instance, Toshima and co-workers (J.Am.Chem.Soc., 1995,117,4822; J.Chem.Soc.,Chem.Commun.,1993,1525; J.Chem.Soc., Chem.Commun.,1992,1306; Heterocycles, 1997,45,851) have synthesised DNA-cleaving hybrid molecules of Formula C below containing enediyne and DNA intercalators. 
DNA cleavage takes place via collapse of the enediyne moiety to a highly reactive diradical which abstracts hydrogen atoms from C4xe2x80x2 of the deoxyribose in the DNA.
The best known anti-cancer agent, which acts by cleaving DNA through generation of active oxygen species, is bleomycin (BLM). It is a glycopeptide which binds to DNA via intercalation of the bis-thiazole group. The compound sequesters iron to form a planar BLM-iron(II) complex and activation by oxygen then provides perferryl capable of abstracting hydrogen atoms from C4xe2x80x2 of deoxyribose. DNA cleavage takes place mainly at the C or T position in GC or GT array.
Examples of hydroxyl radical participation in DNA cleavage are also observed in many other anticancer drugs.
It has how been discovered that artemisinin and synthetic trioxane derivatives can be chemically modified by the attachment of a DNA-binding group to form analogues of artemisinin and synthetic trioxane derivatives which are capable of targeting DNA in pathogenic organisms. Moreover, in the course of synthesising such compounds, other artemisinin and synthetic trioxane derivatives were prepared which do not contain a DNA-binding group but which were found to exhibit cytotoxic and antitumour activity. According to a first aspect of the present invention there is therefore provided a compound of the general formula I 
or a salt thereof,
in which
X represents a hydrogen atom or a group xe2x80x94NR1R2, xe2x80x94CHR8R9 or Ar;
Y represents a hydrogen or halogen atom, an or hydroxyl oxo group, an optionally substituted cycloalkyl, aryl, C-linked heteroaryl or heterocyclylalkyl group or a group xe2x80x94NR3R4, xe2x80x94Oxe2x80x94COxe2x80x94R5 or xe2x80x94OR6; and
Z represents an oxygen atom or a group =NR7; where
R1 and R2 independently represent an optionally substituted alkyl, cycloalkyl, aryl or aralkyl group;
or R1 and R2 together with the interjacent nitrogen atom represent an optionally substituted heterocyclic group or an amino group derived from an optionally substituted amino acid ester;
R3 represents a hydrogen atom or an optionally substituted alkyl, alkenyl or alkynyl group;
R4 represents an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl group;
or R3 and R4 together with the interjacent nitrogen atom represent an optionally substituted heterocyclic group or an amino group derived from an optionally substituted amino acid ester;
R5 represents an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclic or polycyclic group;
R6 represents an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclic or polycyclic group;
R7 represents a hydrogen atom or an optionally substituted alkyl, alkenyl, alkynyl, aralkyl or heterocyclylalkyl group;
R8 represents a hydrogen atom or an optionally substituted alkyl, alkenyl, alkynyl, aryl or alkoxycarbonyl group;
R9 represents a nitro group of an optionally substituted alkyl, alkenyl, alkynyl, aryl, alkanoyl, aroyl, alkoxycarbonyl or aryloxycarbonyl group; or
R8 and R9 together with the interjacent carbon atom represent an optionally substituted cycloalkyl or polycyclic group; and Ar represents an optionally substituted aryl or heteroaryl group;
with the provisos that
(i) when X is a group xe2x80x94NR1R2, then Y is an oxo group and Z is an oxygen atom;
(ii) when Z is a group =NR7, then X is a hydrogen atom and Y is an oxo group;
(iii) when X is a hydrogen atom and Z is an oxygen atom, then Y is not an oxo, methoxy, ethoxy or 3-carboxypropanoyloxy group;
(iv) when Y is a hydrogen atom or a hydroxyl group, then X is a group xe2x80x94CHR8R9;
for use as a cytotoxic agent.
Suitable salts include acid addition salts and these may be formed by reaction of a suitable compound of formula I with a suitable acid, such as an organic acid or a mineral acid. Acid addition salts formed by reaction with a mineral acid are particularly preferred, especially salts formed by reaction with hydrochloric or hydrobromic acid. Compounds of formula I in which X represents a group xe2x80x94NR1R2 or Y represents a group xe2x80x94NR3R4 where R1, R2, R3 and R4 are as defined above are particularly suitable for the formation of such acid addition salts. Suitable salts also include metal salts of compounds in which the substituent R5 or R6 bears a terminal carboxyl group. Such metal salts are preferably formed with an alkali metal atom, such as a lithium, sodium or potassium atom, or with a group xe2x80x94LHal, where L is an alkaline earth metal atom, such as magnesium, and Hal is a halogen atom, preferably a chlorine, bromine or iodine atom. Sodium salts are particularly preferred.
Any alkyl, alkenyl or alkynyl group, unless otherwise specified, may be linear or branched and may contain up to 12, preferably up to 6, and especially up to 4 carbon atoms. Preferred alkyl groups are methyl, ethyl, propyl and butyl. It is preferred that any alkenyl or alkynyl group is not an alk-1-enyl or alk-1-ynyl group. In other words, there should preferably be at least one methylene group xe2x80x94CH2xe2x80x94 or similar sp3-hybridised centre between a carbon atom forming part of the double or triple Cxe2x80x94C bond and the atom to which the group is attached. Preferred alkenyl and alkynyl groups include propenyl, butenyl, propynyl and butynyl groups. When an alkyl moiety forms part of another group, for example the alkyl moiety of an aralkyl group, it is preferred that it contains up to 6, especially up to 4, carbon atoms. Preferred alkyl moieties are methyl and ethyl.
An aryl group may be any monocyclic or polycyclic aromatic hydrocarbon group and may contain from 6 to 24, preferably 6 to 18, more preferably 6 to 16, and especially 6 to 14, carbon atoms. Preferred aryl groups include phenyl, naphthyl, anthryl, phenanthryl and pyryl groups, especially a phenyl or naphthyl, and particularly a phenyl, group. When an aryl moiety forms part of another group, for example the aryl moiety of an aralkyl group, it is preferred that it is a phenyl, naphthyl, anthryl, phenanthryl or pyryl, especially phenyl or naphthyl, and particularly a phenyl, moiety.
An aralkyl group may be an alkyl group substituted by an aryl group. A preferred aralkyl group contains from 7 to 30, particularly 7 to 24 and especially 7 to 18, carbon atoms, particularly preferred aralkyl groups being benzyl, naphthylmethyl, anthrylmethyl, phenanthrylmethyl and pyrylmethyl groups. A particularly preferred aralkyl group is a benzyl group.
A cycloalkyl group may be any saturated cyclic hydrocarbon group and may contain from 3 to 12, preferably 3 to 8, and especially 3 to 6, carbon atoms. Preferred cycloalkyl groups are cyclopropyl, cyclopentyl and cyclohexyl groups.
A polycyclic group may be any saturated or partially unsaturated hydrocarbon group which contains more than one ring system. Such ring systems may be xe2x80x9cfusedxe2x80x9d, that is, adjacent rings have two adjacent carbon atoms in common, xe2x80x9cbridgedxe2x80x9d, that is, the rings are defined by at least two common carbon atoms (bridgeheads) and at least three acyclic chains (bridges) connecting the common carbon atoms, or xe2x80x9cspiroxe2x80x9d compounds, that is, adjacent rings are linked by a single common carbon atom. It is also envisaged that a polycyclic group may contain more than one of these types of ring system. Polycyclic groups preferably contain from 4 to 30, particularly 4 to 26, and especially 6 to 18, carbon atoms. Bicyclic, tricyclic and tetracyclic groups are particularly preferred. Preferred tricyclic groups contain from 4to 14, especially 6 to 10, carbon atoms with bornyl and particularly, isobornyl groups being especially preferred. Preferred tricyclic groups contain from 5 to 20, especially 6 to 14, carbon atoms with adamantyl groups being especially preferred. Preferred tetracyclic groups contain from 6 to 26, especially 6 to 18, carbon atoms. Cholestanyl and cholestenyl groups are further preferred polycyclic groups.
A heteroaryl group may be any aromatic monocyclic or polycyclic ring system which contains at least one heteroatom. Preferably a 5- to 14-membered, and especially a 5- to 10-membered, aromatic ring system containing at least one heteroatom selected from oxygen, sulphur and nitrogen atoms. Preferred heteroaryl groups include pyridyl, pyrylium, thiopyrylium, pyrrolyl, furyl, thienyl, indolinyl, isoindolinyl, indolizinyl, imidazolyl, pyridonyl, pyronyl, pyrimidinyl, pyrazinyl, oxazolyl, thiazolyl, purinyl, quinolinyl, isoquinolinyl, quinoxalinyl, pyridazinyl, benzofuranyl, benzoxazolyl and acridinyl groups. A C-linked heteroaryl group is therefore a heteroaryl group as defined above which is linked to the tetracyclic 1,2,4-trioxane moiety of a compound of general formula I via a carbon atom in the heteroaromatic ring system.
A heterocyclic group may be any monocyclic or polycyclic ring system which contains at least one heteroatom and may be unsaturated or partially or fully saturated. The term xe2x80x9cheterocyclicxe2x80x9d thus includes heteroaryl groups as defined above as well as non-aromatic heterocyclic groups. Preferably, a heterocyclic group is a 3- to 18- membered, particularly a 3- to 14-membered, especially a 5- to 10-membered, ring system containing at least one heteroatom selected from oxygen, sulphur and nitrogen atoms. Preferred heterocyclic groups include the specific heteroaryl groups named above as well as pyranyl, piperidinyl, pyrrolidinyl, dioxanyl, piperazinyl, morpholinyl, thiomorpholinyl, morpholinosulphonyl, tetrahydroisoquinolinyl and tetrahydrofuranyl groups.
A heterocyclylalkyl group may be an alkyl group substituted by a heterocyclic group. Preferably, the heterocyclic moiety is a 3- to 18- membered, particularly a 3- to 14-membered, and especially a 5- to 10-membered, heterocyclic group as defined above and the alkyl moiety is a C1-6 alkyl, preferably C1-4 alkyl, and especially methyl, group.
An amino acid may be any xcex1-amino acid, such as glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, aspartic acid, glutamic acid, aspargine, glutamine, lysine, hydroxylysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline or phenylglycine, and includes both D- and L-configurations. An amino acid ester may be any ester of such an amino acid, alkyl esters, particularly C1-4 alkyl esters, being especially preferred.
When any of the foregoing substituents are designated as being optionally substituted, the substituent groups which are optionally present may be any one or more of those customarily employed in the development of pharmaceutical compounds and/or the modification of such compounds to influence their structure/activity, stability, bioavailability or other property. Specific examples of such substituents include, for example, halogen atoms, nitro, cyano, hydroxyl, cycloalkyl, alkyl, alkenyl, haloalkyl, cycloalkyloxy, alkoxy, haloalkoxy, amino, alkylamino, dialkylamino, formyl, alkoxycarbonyl, carboxyl, alkanoyl, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylsulphonato, arylsulphinyl, arylsulphonyl, arylsulphonato, carbamoyl, alkylamido, aryl, aralkyl, optionally substituted aryl, heterocyclic and alkyl- or aryl-substituted heterocyclic groups. When any of the foregoing substituents represents or contains an alkyl or alkenyl substituent group, this may be linear or branched and may contain up to 12, preferably up to 6, and especially up to 4, carbon atoms. A cycloalkyl group may contain from 3 to 8, preferably from 3 to 6, carbon atoms. An aryl group or moiety may contain from 6 to 10 carbon atoms, phenyl groups being especially preferred. A heterocyclic group or moiety may be a 5- to 10-membered ring system as defined above. A halogen atom may be a fluorine, chlorine, bromine or iodine atom and any group which contains a halo moiety, such as a haloalkyl group, may thus contain any one or more of these halogen atoms.
It is particularly preferred that X represents a hydrogen atom or a group xe2x80x94NR1R2 or CHR8R9, where R1, R2, R8 and R9 are as defined above.
Preferably, X represents a hydrogen atom. It is also preferred that Z represents an oxygen atom.
In one aspect, it is preferred that Y represents a halogen atom, particularly a fluorine or bromine, and especially a fluorine, atom.
In another preferred aspect Y may represent a C3-8 cycloalkyl group, a C6-18 aryl group, a 5- to 10-membered C-linked heteroaryl group or a 5- to 10-membered heterocyclyl-C1-6 alkyl group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-4 alkyl, C2-4 alkenyl, C1-4 haloalkyl, C1-4 alkoxy, amino, C1-4 alkylamino, di(C1-4 alkyl)amino, carboxyl, C6-10 aryl, 5 to 10--membered heterocyclic and C1-4 alkyl- or phenyl-substituted 5- to 10-membered heterocyclic groups. Preferably Y represents a C6-18 aryl group optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-4 alkyl, C2-4 alkenyl, C1-4 haloalkyl, C1-4 alkoxy, C1-4 haloalkoxy, amino, C1-4 alkylamino, di (C1-4 alkyl)amino and carboxyl groups. In particular, Y may represent a phenyl, naphthyl, anthryl or phenanthryl group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms and hydroxyl, methyl, vinyl, C1-4 alkoxy and carboxyl groups.
In a particularly preferred sub-group of compounds, Y represents a phenyl, fluorophenyl, chlorophenyl, bromophenyl, trimethylphenyl, vinylphenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl, carboxylphenyl, naphthyl, hydroxynaphthyl, methoxynaphthyl, anthryl or phenanthryl group. Compounds in which Y represents a dimethoxyphenyl or trimethoxyphenyl group are especially preferred.
In a further preferred aspect, Y may represent a group xe2x80x94NR3R4 where R3 represents a hydrogen atom or a C1-6 alkyl group and R4 represents a C1-6 alkyl, C3-8 cycloalkyl, C6-10 aryl or C7-16 aralkyl group, or R3 and R4 together with the interjacent nitrogen atom represent a 5- to 10-membered heterocyclic group or an amino group derived from a C1-6 alkyl ester of an amino acid, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C1-4 haloalkyl, C1-6 alkoxycarbonyl, phenyl, halophenyl, C1-4 alkylphenyl, C1-4 haloalkylphenyl, C1-4 alkoxyphenyl, benzyl, pyridyl and pyrimidinyl groups. In particular, Y may represent a group xe2x80x94NR3R4 where R3 represents a hydrogen atom or a C1-4 alkyl group and R4 represents a C1-4 alkyl, C3-6 cycloalkyl, phenyl or benzyl group, or R3 and R4 together with the interjacent nitrogen atom represent a 6- to 10-membered heterocyclic group or an amino group derived from a C1-4 alkyl ester of an amino acid, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 haloalkyl, C1-4 alkoxycarbonyl, phenyl, halophenyl, C1-4 alkylphenyl, C1-4 haloalkylphenyl, C1-4 alkoxyphenyl, benzyl, pyridyl and pyrimidinyl groups.
In a particularly preferred sub-group of these compounds, Y represents a propylamino, cyclopentylamino, cyclohexylamino, phenylamino, fluorophenylamino, chlorophenylamino, bromophenylamino, iodophenylamino, methoxycarbonylphenylamino, biphenylamino, benzylamino, fluorobenzylamino, bis(trifluoromethyl)-benzylamino, phenylethylamino, phenylmethoxycarbonyl-methylamino, diethylamino, morpholinyl, thiomorpholinyl, S,S-dioxothiomorpholinyl (morpholinosulphonyl), indolinyl, tetrahydroisoquinolinyl, phenylpiperazinyl, fluorophenylpiperazinyl, chlorophenylpiperazinyl, methylphenylpiperazinyl, trifluoromethylphenylpiperazinyl, methoxyphenylpiperazinyl, benzylpiperazinyl, pyridylpiperazinyl and pyrimidinylpiperazinyl group. Compounds in which Y represents a phenylamino or fluorophenylamino group are especially preferred.
In another preferred aspect, Y may represent a group xe2x80x94Oxe2x80x94COxe2x80x94R5 where R5 represents an optionally substituted alkyl, aryl, aralkyl, heterocyclic or polycyclic group. Preferably, R5 represents a C1-6 alkyl, C6-18 aryl, 5- to 18- membered heterocyclic or C4-26 polycyclic group, each group being optionally substituted by one or more substituents selected form the group consisting of halogen atoms, hydroxyl, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy, C1-4 haloalkoxy, oxo, carboxyl and carboxylato groups. More preferably, R5 represents a C1-4 alkyl, C6-14 aryl, 5- to 14- membered heterocyclic or C6-14 polycyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-4 alkyl, C1-4 haloalkyl and oxo groups.
In a particularly preferred sub-group of these compounds, R5 represents a methyl, phenyl, hydroxynaphthyl, anthryl, anthraquinonyl, quinolinyl, isoquinolinyl, quinoxalinyl or acridinyl group.
In a further preferred aspect, Y may represent a group xe2x80x94OR6 where R6 represents an optionally substituted alkyl, aryl, aralkyl, heterocyclic or polycyclic group. Preferably, R6 represents a C1-6 alkyl, C6-24 aryl, C7-30 aralkyl, 5- to 18 - membered heterocyclic or C4-26 polycyclic group, each group being optionally substituted by one or more substituents selected form the group consisting of halogen atoms, hydroxyl, C1-8 alkyl, C1-4 haloalkyl, C1-4 alkoxy, C1-4 haloalkoxy, carboxyl and C1-4 alkoxycarbonyl groups. More preferably, R6 represents a C1-4 alkyl, C6-14 aryl, C7-18 aralkyl, 5- to 14-membered heterocyclic or C6-18 polycyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy and C1-4 haloalkoxy groups.
In a particularly preferred sub-group of these compounds, R6 represents a trifluoroethyl, methoxyphenyl, naphthyl, benzyl, fluorobenzyl, naphthylmethyl, anthrylmethyl, phenanthrylmethyl, pyrylmethyl, quinolinyl, trifluoromethylquinolinyl or cholestenyl group. Compounds in which R6 represents a naphthylmethyl group are particularly preferred.
In another aspect, it is preferred that Z represents a group =NR7. Preferably, R7 represents a hydrogen atom or a C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C7-16 aralkyl or 5- to 10-membered-heterocyclic-C1-6 alkyl group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, cyano, nitro, C1-4 haloalkyl, formyl, C1-6 alkoxycarbonyl, C1-6 alkanoyl, phenylsulphinyl, phenylsulphonyl and phenylsulphonato groups.
It is also preferred that R7 represents a hydrogen atom or a C1-4 alkyl, C2-4 alkenyl, C7-11 aralkyl or 5- to 10-membered-heterocyclic-C1-4 alkyl group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl and formyl groups.
Preferably, R7 represents a hydrogen atom or a C1-4 alkyl or benzyl group, each group being optionally substituted by one or more halogen atoms or hydroxyl groups.
More preferably, R7 represents a hydrogen atom or a methyl, butyl, formylmethyl, hydroxyethyl, propenyl, benzyl, halobenzyl, especially fluorobenzyl, pyridylmethyl, thienylmethyl or furylmethyl group.
In a particularly preferred sub-group of these compounds, R7 represents a hydrogen atom or a benzyl or fluorobenzyl group.
In another preferred aspect, X may represent a group xe2x80x94CHR8R9 where R8 represents a hydrogen atom or a C1-6 alkyl or C1-6 alkoxycarbonyl group and R9 represents a nitro, C1-6 alkyl, C1-6 alkanoyl, C7-11 aroyl, C1-6 alkoxycarbonyl or C6-10 aryloxycarbonyl group, or R8 and R9 together with the interjacent carbon atom represent a C3-8 cycloalkyl or C4-26 polycyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy and C1-4 haloalkoxy groups. Preferably, X represents a group xe2x80x94CHRR9 where R8 represents a hydrogen atom or a C1-4 alkyl or C1-4 alkoxycarbonyl group and R9 represents a nitro, C1-4 alkyl, C1-4 alkanoyl, benzoyl, C1-4 alkoxycarbonyl or benzoxycarbonyl group, or R3 and R4 together with the interjacent carbon atom represent a C3-6 cycloalkyl, C6-10 bicyclic or C6-14 tricyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-4 alkyl and C1-4 haloalkyl groups.
In a particularly preferred sub-group of these compounds, X represents a nitromethyl, isopropyl, methoxycarbonylethyl, ethoxycarbonylmethyl, di(ethoxycarbonyl)methyl, benzoylmethyl, cyclohexyl or adamantyl group. Compounds in which X represents a nitromethyl, isopropyl, methoxycarbonylethyl, ethoxycarbonylmethyl, di(ethoxycarbonyl)methyl or benzoylmethyl group are especially preferred.
In another preferred aspect, X represents a C6-18 aryl or 5- to 18- membered heteroaryl group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C-4 haloalky, C1-4 alkoxy, di(C1-4alkyl)amino, C7-10 aralkyl and heterocyclic groups. Preferably, X represents a C6-10 aryl group optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C1-4 alkoxy, di(C1-4 alkyl)amino and heterocyclic groups.
In a particularly preferred sub-group of compounds, X represents a phenyl, chlorophenyl or bromophenyl group.
IN a further preferred aspect, X may represent a group xe2x80x94NR1R2 where R1 and R2 independently represent a C1-6 alkyl, C6-10 aryl or C7-16 aralkyl group, or R1 and R2 together with the interjacent nitrogen atom represent a 3- to 14-membered heterocyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C1-4 haloalkyl and C1-6 alkoxycarbonyl groups. Preferably, X represents a group xe2x80x94NR1R2 where R1 and R2 independently represent a C1-4 alkyl or C7-10 aralkyl group, or R1 and R2 together with the interjacent nitrogen atom represent a 5- to 10- membered heterocyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C1-4 haloalkyl and C1-4 alkoxycarbonyl groups.
In a particularly preferred sub-group of compounds, X represents a dibenzylamino or indolinyl group.
The present invention also provides the use of a compound of the general formula I as defined above for the manufacture of a medicament for use as a cytotoxic agent.
A compound of the general formula I as defined above for use as an antitumour agent or for use in the treatment of cancer is also provided. In addition, the present invention provides the use of a compound of the general formula I as defined above for the manufacture of a medicament for use as an antitumour agent or for use in the treatment of cancer.
Some of the compounds of formula I are already known. specifically, artemisinin derivatives are known in which the oxygen atom at the 11-position has been replaced by a nitrogen atom to produce 11-azaartemisinin and N-substituted derivatives thereof. For instance, Torok and Ziffer (J.Med.Chem.,1995,38, 5045-5050) synthesised 11-azaartemisinin and derivatives thereof in which the 11 -nitrogen atom is substituted by a methyl, isobutyl, allyl, formylmethyl, benzyl, pyrid-2-ylmethyl, 2-thio-phenemethyl or furfuryl group. These compounds were tested for in vitro activity against a chloroquine-resistant strain of Plasmodium falciparum and all compounds except the N-benzyl derivatives exhibited some activity. Mekonnen and Ziffer (Tetrahedron Letters,1997,38(5), 731-734) also synthesized 11-azaartemisinin and its N-allyl and N-formylmethyl derivatives and described the conversion of 11-azaartemisinin by a Michael addition reaction to derivatives in which the 11-nitrogen atom is substituted by a cyanoethyl, ethoxycarbonylethyl, methylcarbonylethyl, phenylsulphinylethyl, phenylsulphonylethyl or phenylsulphonatoethyl group. However, these derivatives were not tested for biological activity.
Artemisinin derivatives are also known in which the oxygen atom at C-10 has been replaced by an amine group. For instance, Yang et al (Biorg. Med. Chem. Lett., 1995, 5, 1791-1794) synthesised ten new artemisinin derivatives in which the oxygen atom at C-10 was replaced by a group xe2x80x94NHAr where Ar represents a phenyl, 3-chlorophenyl, 4-chlorophenyl, 3-bromophenyl, 4-bromophenyl, 4-iodophenyl, 4-methylphenyl, 4-methoxyphenyl, 3-carboxylphenyl or 4-carboxylphenyl group. These compounds were tested for in vivo activity against the K173 strain of Plasmodium berghei and found to be active.
Further artemisinin derivatives are also known in which one of the hydrogen atoms in the methyl group attached to the C-9 carbon atom in artemisinin, that is, one of the hydrogen atoms attached to the C-16 carbon atom, has been replaced by a sulphur-, nitrogen- or carbon-linked group. For instance, Paitayatat et al (J.Med.Chem.,1997,40,633-638) synthesised, inter alia, two new artemisinin derivatives in which the C-16 carbon atom is substituted by a phenylthio or a imidazol-1-yl group and demonstrated that these compounds are active against Plasmodium falciparum. Avery et al (J.Med.Chem.,1996,39,4149-4155) synthesised artemisinin derivatives in which the C-16 carbon atom is substituted by a methyl, ethyl, n-propyl, benzyl or 4-chlorobenzyl group and the five corresponding 10-deoxo derivatives. Moreover, the activity of the five 10-deoxo derivatives against Plasmodium falciparum was also demonstrated. U.S. Pat. No. 5,216,175 also specifically discloses artemisinin derivatives in which the C-16 carbon atom of artemisinin is substituted by a methyl, isopropyl, n-butyl, n-dodecyl or benzyl group and demonstrates activity for these compounds against Plasmodium falciparum. 
Artemisinin derivatives are also known in which the oxygen atom at C-10 forms part of a variety of ester groups. For instance, Li et al (Acta Pharmaceutical Sinica,1981,16,429) synthesised, inter alia, ester derivatives of artemisinin in which the C-10 carbon atoms is substituted by a group of formula xe2x80x94Oxe2x80x94COxe2x80x94RA where RA represents a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, phenyl 4-methylphenyl, 4-methoxyphenyl, 3,4,5-trimethoxyphenyl, benzyl or styryl group and evaluated these compounds for activity against a chloroquine-resistant strain of Plasmodium berghei. Cao et al (Nanjing Yaoxueyun Xuebao, 1982,1,53) also synthesised ester derivatives of artemisinin in which the C-10 carbon atom is substituted by a group of formula xe2x80x94Oxe2x80x94COxe2x80x94RB where RB represents an n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-undecyl, 1-(propyl)butyl, 1-chloroethyl or t-butyl group and tested these compounds for suppressive antimalarial activity against Plasmodium berghei in mice.
Further artemisinin derivatives are known in which the oxygen atom at C-10 forms part of a variety of ether groups. For instance, Li et al (Acta Pharmaceutical Sinica,1981,16,429) synthesised, inter alia, ether derivatives of artemisinin in which the C-10 carbon atoms is substituted by a group of formula xe2x80x94Oxe2x80x94RB where RB represents a methyl, ethyl, n-propyl, isopropyl, n-butyl, isopentyl, tert-pentyl, n-octyl, 2-hydroxyethyl, 2-methoxyethyl, prop-2-enyl, 3-phenylprop-2-enyl, cyclopentyl, cyclohexyl, benzyl or 4-methoxybenzyl group and evaluated these compounds for activity against a chloroquine-resistant strain of Plasmodium berghei. Ramu and Baker (J.Med.Chem.1995, 38,1911-1921) synthesised glucuronide tri-ethanoyloxy ester derivatives of dihydroartemisinin and hydrolysed such derivatives to form the xcex1- and xcex2-dihydroartemisininglucuronides. The latter compounds exhibited in vitro activity against Plasmodium falciparum. Vishwakarma et al (J.Nat.Prod.,1992, 55(8),1142-1144) described a stereoselective synthesis of xcex1-artelinic acid and its methyl ester and sodium salt and tested the acid for antimalarial activity against Plasmodium knowlesi in vivo. This article also refers to xcex2-artelinic acid and its potassium salt. Lin et al (J.Med.Chem.,1990,33,2610-2614) referred to their earlier synthesis of xcex2-artelinic acid and its sodium salt and the antimalarial activity of these compounds in mice infected with Plasmodium berghei. 
U.S. Pat. No. 5,486,535 describes the activity of dehydroartemisinin and ether derivatives of artemisinin in which the C-10 carbon atom is substituted by a group of formula xe2x80x94Oxe2x80x94RC where RC represents a methyl, ethyl, iso-propyl, iso-butyl or see-butyl group against Toxoplasma gondii. 
U.S. Pat. No. 5,225,427 discloses certain 10-substituted ether derivatives of artemisinin in which the C-10 carbon atoms is substituted by a group of formula xe2x80x94Oxe2x80x94RD where RD represents a 1-ethanoylethyl, 1,3-bis(isopropoxy)prop-2-yl, but-3-yn-2-yl, 2-methylbut-3-yn-2yl, 2-ethylbut-3-yn-2-yl, 2-(4-chlorophthalimido)ethyl, 3-(4-carboxylphenyl)-isoxazol-5-ylmethyl, 3-chloroisoxazol-5-ylmethyl or 3-bromoisoxazol-5-yl methyl group. The activity of all these compounds against Plasmodium berghei was demonstrated in vivo and the 2-ethylbut-3-yn-2-yl and 3-(4-carboxylphenyl)isoxazol-5-ylmethyl derivatives were also shown to possess antiamoebic activity against Entamoeba histolytica in vitro.
Certain compounds of the general formula I are novel and the invention therefore further provides a compound of the general formula I as defined above with the further provisos that
(i) when X represents a group xe2x80x94NR1R2, then R1 and R2 together with the interjacent nitrogen atom do not represent an imidazol-1-yl group;
(ii) when Y represents a group xe2x80x94NR3R4 and R4 represents a phenyl, 3-chlorophenyl, 4-chlorophenyl, 3-bromophenyl, 4-bromophenyl, 4-iodophenyl, 4-methylphenyl, 4-methoxyphenyl, 3-carboxylphenyl or 4-carboxylphenyl group, then R3 is an optionally substituted alkyl group;
(iii) when Y represents a group xe2x80x94Oxe2x80x94COxe2x80x94R5, R5 does not represent a methyl, ethyl, propyl, butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-undecyl, 1-(propyl)butyl, 1-chloroethyl, phenyl, 4-methylphenyl, 4-methoxyphenyl, 3,4,5-trimethoxyphenyl, benzyl or styryl group;
(iv) when Y represents a group xe2x80x94ORxe2x80x2, R6 does not represent an n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, iso-pentyl, tert-pentyl, n-octyl, 2-hydroxyethyl, 2-methoxyethyl, 1-ethanoylethyl, 1,3-bis(isopropoxy)prop-2-yl, prop-2-enyl, 3-phenylprop-2-enyl, but-3-yn-2-yl, 2-methylbut-3-yn-2-yl, 2-ethylbut-3-yn-2-yl, cyclopentyl, cyclohexyl, benzyl, 4-methoxybenzyl, 4-(methoxycarbonyl)benzyl, 4-carboxylphenyl, 4-(sodium carboxylato)benzyl, 4-(potassium carboxylato)benzyl, 3-chloroisoxazol-5-ylmethyl, 3-bromoisoxazol-5-ylmethyl, 3-(4-carboxylphenyl)isoxazol-5-ylmethyl, 2-(4-chlorophthalimide)ethyl, glucuronide or triethanoyloxyglucuronide group,
(v) when Z is a group +50 NEC, R7 does not represent a hydrogen atom or a methyl, isobutyl, allyl, formylmethyl, pyrid-2-ylmethyl, thenyl, furfuryl, benzyl, cyanoethyl, ethoxycarbonylethyl, ethanoylethyl, phenylsulphinylethyl, phenylsulphonylethyl or phenylsulphonatoethyl group;
(vi) when X represents an n-pentyl, n-tridecyl or 2-methylpropyl group, then Y represents a hydrogen or hydroxyl group; and
(vii) when X represents a group xe2x80x94CHR8R9 where R8 represents a hydrogen atom, then R9 does not represent a methyl, ethyl, n-propyl, benzyl or 4-chlorobenzyl group.
It should also be appreciated that the compounds of general formula I are capable of existing as different geometric and optical isomers. The present invention thus includes both the individual isomers and mixtures of such isomers.
The present invention also provides processes for the preparation of novel compounds of the general formula I as defined in the ante-preceding paragraph. For instance, compounds of general formula I in which X represents a hydrogen atom, Y represents a halogen atom, an optionally substituted cycloalkyl, aryl, C-linked heteroaryl or heterocyclylalkyl group or a group xe2x80x94NR3R4, where R3 and R4 are as defined above, and Z represents an oxygen atom may be prepared by reacting a compound of the general formula II 
in which Q represents a hydrogen atom or trimethylsilyl group, with a suitable halogenating agent to form a compound of the general formula I in which Y represents a halogen atom; and, if desired, reacting the compound of general formula I thus formed either with a Grignard reagent of the general formula YMgHal where Y is an optionally substituted cycloalkyl, aryl, C-linked heteroaryl or heterocyclylalkyl group and Hal is a halogen atom to form a compound of general formula I in which Y represents an optionally substituted cycloalkyl, aryl, C-linked heteroaryl or heterocyclylalkyl group or with an amine of the general formula HNR3R4 where R3 and R4 are as defined above to form a compound of general formula I in which Y represents a group xe2x80x94NR3R4 where R3 and R4 are as defined above.
Suitable halogenating agents for forming compounds of the general formula I in which Y represents a halogen atom include diethylaminosulphur trifluoride, chlorotrimethylsilane, bromotrimethylsilane and iodotrimethylsilane. In particular, compounds of the general formula I in which Y represents a chlorine, bromine or iodine atom may be prepared by reacting a compound of the general formula II in which Q represents a trimethylsilyl group with a suitable chlorinating, brominating or iodinating agent respectively, such as chlorotrimethlysilane, bromotrimethylsilane or iodotrimethylsilane respectively. This reaction may be conveniently carried out in the presence of a solvent. Suitable solvents include halogenated hydrocarbons, especially chlorinated hydrocarbons, such as dichloromethane. Preferably, the reaction is carried out at a temperature of xe2x88x9230xc2x0 C. to +10xc2x0, particularly xe2x88x925xc2x0 C. to +5xc2x0 C., about 0xc2x0 C. being especially preferred.
Compounds of the general formula I in which Y represents a fluorine atom may be conveniently prepared by reacting a compound of the general formula II in which Q represents a hydrogen atom with a suitable fluorinating agent, such as diethylaminosulphur trifluoride. This reaction may be conveniently carried out in the presence of a solvent, suitable solvents including halogenated hydrocarbons, especially chlorinated hydrocarbons, such as dichloromethane. Preferably, the reaction is carried out at xe2x88x925xc2x0 C. to room temperature, that is, xe2x88x925 to +35xc2x0 C., preferably 0 to 30xc2x0 C. The reaction may also be carried out under an inert atmosphere, such as nitrogen.
Suitable Grignard reagents for forming compounds of the general formula I in which Y is an optionally substituted cycloalkyl, aryl, C-linked heteroaryl or heterocyclylalkyl group include compounds of the general formula YMgHal where Hal represents a chlorine, bromine or iodine atom. However, it is particularly preferred that Hal represents a bromine atom. The reaction of a compound of the general formula I in which Y represents a halogen, preferably a bromine, atom with a Grignard reagent may be conveniently carried out in the presence of a solvent. Suitable solvents include ethers, such as diethyl ether. Preferably, the reaction is carried out under an inert atmosphere, such as nitrogen, at a temperature of xe2x88x925xc2x0 C. to +5xc2x0 C., 0xc2x0 C. being especially preferred. This method produces a single pure isomer of the final product.
The reaction of an amine with a compound of the general formula I in which Y represents a halogen, preferably a bromine, atom to form a compound of the general formula I in which Y represents a group xe2x80x94NR3R4 where R3 and R4 are as defined above may be conveniently carried out in the presence of a solvent. Suitable solvents include halogenated hydrocarbons, especially chlorinated hydrocarbons, such as dichloromethane, and ethers, such as tetrahydrofuran. Preferably, the reaction is carried out at a temperature of xe2x88x925xc2x0 C. to +5xc2x0 C., 0xc2x0 C. being especially preferred.
When a compound of the general formula I in which Y represents a bromine atom is to be further reacted with a Grignard reagent or an amine to form a compound of the general formula I in which Y represents an optionally substituted cycloalkyl, aryl, C-linked heteroaryl or heterocyclylalkyl group or a group xe2x80x94NR3R4 where R3 and R4 are as defined above, it is preferred that the compound of the general formula I in which Y represents a bromine atom is generated in situ by reacting a compound of the general formula II in which Q represents a trimethylsilyl group with bromotrimethylsilane.
A compound of the general formula II in which Q represents a trimethylsilyl group may be prepared by reacting dihydroartemisinin, that is, the compound of general formula II in which Q represents a hydrogen atom, with chlorotrimethylsilane in the presence of a base, such as pyridine or triethylamine. Preferably, the reaction is carried out at room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
Dihydroartemisinin, that is, the compound of general formula II in which Q represents a hydrogen atom, is a known compound and can be prepared by known processes.
Compounds of the general formula I in which Y represents an optionally substituted cycloalkyl, aryl, C-linked heteroaryl or heterocyclylalkyl group can also be prepared by reacting 9,10-anhydroartemisinin with a compound of the general formula Yxe2x80x94H, where Y is as defined above, in the presence of a suitable Lewis acid. This method produces a mixture of isomers in the final product.
Suitable Lewis acids include boron trifluoride diethyl etherate and trifluoromethanesulphonic acid. The reaction may be conveniently carried out in the presence of a solvent. Suitable solvents include halogenated hydrocarbons, especially chlorinated hydrocarbons, such as dichloromethane. Preferably, the reaction is carried out under an inert atmosphere, such as nitrogen, at room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
9,10-Anhydroartemisinin may be conveniently prepared by reacting dihydroartemisinin with trifluoroacetic anhydride. The reaction may be conveniently carried out in the presence of a solvent, preferably a halogenated hydrocarbon, and especially a chlorinated hydrocarbon, such as dichloromethane. It is also preferred that the reaction is carried out in the presence of a base, such as pyridine or a derivative thereof, for example, dimethylaminopyridine. Preferably, the reaction is carried out under an inert atmosphere, such as nitrogen, at a temperature of xe2x88x925xc2x0 C. to +5xc2x0 C., preferably 0xc2x0 C., with the reaction mixture being subsequently allowed to warm to room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
Compounds of the general formula I in which Y represents an optionally substituted aryl or C-linked heteroaryl group can also be prepared by reacting 10-trichloroacetimidoyl-10-deoxoartemisinin with a compound of the general formula Yxe2x80x94H, where Y is as defined above, in the presence of a suitable Lewis acid, such as boron trifluoride diethyl etherate. It is preferred that the 10-trichloroacetimidoyl-10-deoxoartemisinin is generated in situ by reacting a compound of the general formula II in which Q represents a hydrogen atom with trichloroacetonitrile in the presence of a suitable base, such as 1,8-diazabicyclo[5.4.0] undecane. Preferably, the reaction to form 10-trichloroacetimidoyl-10-deoxoartemisinin is carried out at room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C. The reaction may be conveniently carried out in the presence of a solvent Suitable solvents include halogenated hydrocarbons; especially chlorinated hydrocarbons, such as dichloromethane. Preferably, the remainder of the reaction is carried out under an inert atmosphere, such as nitrogen. Preferably, the remainder of the reaction is carried out at a temperature of xe2x88x9260 to xe2x88x9220xc2x0 C., particularly xe2x88x9255 to xe2x88x9230xc2x0 C., and especially xe2x88x9240 to xe2x88x9250xc2x0 C.
Compounds of the general formula I in which Y represents an optionally substituted aryl or C-linked heteroaryl group can also be prepared by reacting a 10-acyloxyartemisinin compound in which the acyloxy group is of formula A(Cxe2x95x90O)xe2x80x94Oxe2x80x94, where A represents an optionally substituted alkyl, cycloalkyl, aryl, aralkyl, heterocyclic or polycyclic group, with a compound of the general formula Yxe2x80x94H, where Y is as defined above, in the presence of a suitable Lewis acid. Suitable Lewis acids include boron trifluoride diethyl etherate, tin(IV) chloride, copper(II)-trifluoromethanesulfonate and trifluoromethane-sulphonic acid. It is preferred that the Lewis acid is boron trifluoride diethyl etherate.
When A represents an optionally substituted alkyl group, unless otherwise specified, this may be linear or branched and may contain up to 12, preferably up to 6, and especially up to 4 carbon atoms. Preferred alkyl groups are methyl, ethyl, propyl and butyl.
When A represents an optionally substituted aryl group, this may be any aromatic hydrocarbon group and may contain from 6 to 24, preferably 6 to 18, more preferably 6 to 16, and especially 6 to 14, carbon atoms. Preferred aryl groups include phenyl, naphthyl, anthryl, phenanthryl and pyryl groups, especially phenyl, naphthyl and anthryl groups. When an aryl moiety forms part of another group, for example the aryl moiety of an aralkyl group, it is preferred that it is a phenyl, naphthyl, anthryl, phenanthryl or pyryl, especially a phenyl or naphthyl, and particularly a phenyl, moiety.
When A represents an optionally substituted aralkyl group, this may be any alkyl group substituted by an aryl group. A preferred aralkyl group contains from 7 to 30, particularly 7 to 24, more particularly 7 to 18, and especially 7 to 10, carbon atoms, particularly preferred aralkyl groups being benzyl, naphthylmethyl, anthrylmethyl, phenanthrylmethyl and pyrylmethyl groups, a benzyl group being especially preferred.
When A represents an optionally substituted cycloalkyl group, this may be any saturated or partially unsaturated cyclic hydrocarbon group and may contain from 3 to 12, preferably 3 to 8, and especially 3 to 6, carbon atoms. Preferred cycloalkyl groups are cyclopropyl, cyclopentyl and cyclohexyl groups.
When A represents an optionally substituted polycyclic group, this may be any saturated or partially unsaturated hydrocarbon group which contains more than one ring system. Such ring systems may be xe2x80x9cfusedxe2x80x9d, that is, adjacent rings have two adjacent carbon atoms in common, xe2x80x9cbridgedxe2x80x9d, that is, the rings are defined by at least two common carbon atoms (bridgeheads) and at least three acyclic chains (bridges) connecting the common carbon atoms, or xe2x80x9cspiroxe2x80x9d compounds, that is, adjacent rings are linked by a single common carbon atom. It is also envisaged that a polycyclic group may contain more than one of these types of ring system. Polycyclic groups preferably contain from 4 to 30, particularly 4 to 26, and especially 6 to 18, carbon atoms. Bicyclic, tricyclic and tetracyclic groups are particularly preferred. Preferred bicyclic groups contain from 4 to 14, especially 6 to 10, carbon atoms. Preferred tricyclic groups contain from 5 to 20, especially 6 to 14, carbon atoms with anthraquinone groups being especially preferred. Preferred tetracyclic groups contain from 6 to 26, especially 6 to 18, carbon atoms.
Optional substituents for the substituent A may be any of those previously identified as suitable in this respect.
The reaction may be conveniently carried out in the presence of a solvent. Suitable solvents include halogenated hydrocarbons, especially chlorinated hydrocarbons, such as dichloromethane. Preferably, the reaction is carried out under an inert atmosphere, such as nitrogen. Preferably, the reaction is carried out at a temperature of xe2x88x9260 to xe2x88x9220xc2x0 C., particulariy xe2x88x9255 to xe2x88x9230xc2x0 C., and especially xe2x88x9240 to xe2x88x9250xc2x0 C.
Compounds of formula I in which Y represents a substituted aryl group where at least one of the substituents is a hydroxyl group can also be prepared by rearrangement of the corresponding C-10 ether linked artemisinin derivative so that the oxygen atom of the ether link becomes the oxygen atom of the hydroxyl group in the substituted aryl group of the desired product. Such a rearrangement can be effected by reacting the corresponding C-10 ether linked artemisinin derivative with a Lewis acid, such as a boron trifluoride diethyl etherate. The reaction is conveniently carried out in the presence of a solvent such as dichloromethane at a temperature of xe2x88x925xc2x0 C. to 5xc2x0 C., preferably 0xc2x0 C.
Certain compounds of the general formula I may also be prepared by conversion of another compound of general formula I. For instance, 10-(4-vinylphenyl) dihydroartemisinin may be converted to 10-(4-carboxylphenyl)dihydroartemisinin by reaction with an oxidising agent, such as potassium permanganate. Also, compounds of general formula I which contain a heterocyclic moiety having at least one sulphur atom in the ring system may be oxidised to form compounds of general formula I in which the or each sulphur atom has been converted to a sulphinyl or sulphonyl group by reaction with a suitable oxidising agent. Suitable oxidising agents include 4-methylmorpholine N-oxide (NMO), tetrapropylammonium perruthenate (TPAP) and mixtures thereof. The reaction may be conveniently carried out in the presence of a solvent, suitable solvents including halogenated hydrocarbons, especially chlorinated hydrocarbons, such as dichloromethane. Preferably, the reaction is carried out at room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C. The reaction may also be carried out under an inert atmosphere, such as nitrogen.
Compounds of general formula I in which x represents a hydrogen atom, Y represents a group xe2x80x94Oxe2x80x94COxe2x80x94R5, where R5 is as defined above, and Z represents an oxygen atom may be prepared by reacting dihydroartemisinin with a compound of the general formula R5xe2x80x94COxe2x80x94W where W is a halogen atom or hydroxyl group or a group xe2x80x94Oxe2x80x94COxe2x80x94R5 where R5 is as defined above. When W is a halogen atom, it is preferred that the halogen atom is a chlorine atom.
The reaction of dihydroartemisinin with an acid chloride or acid anhydride, that is, compounds of formula R5xe2x80x94COxe2x80x94W in which W represents a chlorine atom or a group xe2x80x94Oxe2x80x94COxe2x80x94R5 respectively, may be conveniently carried out in the presence of a solvent. Suitable solvents include halogenated hydrocarbons, particularly chlorinated hydrocarbons, such as dichloromethane. The reactions may also be carried out in the presence of a base, such as 4-(N,N-dimethylamino)pyridine, under an inert atmosphere, for instance, under nitrogen. Preferably, the reaction is carried out at a temperature of xe2x88x925xc2x0 C. to room temperature. Ideally, the reagents are initially mixed together at 0xc2x0 C. and the reaction mixture is then allowed to warm to room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
The reaction of dihydroartemisinin with a carboxylic acid, that is a compound of formula R5xe2x80x94COxe2x80x94W in which W represents a hydroxyl group, may be conveniently carried out in the presence of a suitable solvent. Suitable solvents include ethers, particularly cyclic ethers, such as tetrahydrofuran. The reaction may also be carried out in the presence of a suitable condensing agent which acts to activate the hydroxyl group of the carboxylic acid for displacement by the hydroxyl group of dihydroartemisinin. A suitable condensing agent is a combination of triphenylphosphine and diethyl azodicarboxylate. The reaction may be carried out under an inert atmosphere, for instance, under nitrogen. Preferably, the reaction is carried out at a temperature of xe2x88x925xc2x0 C. to room temperature, preferably 0xc2x0 C. to room temperature. Ideally, the reagents are initially mixed together at 0xc2x0 C. and the reaction mixture is then allowed to warm to room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 35xc2x0 C.
Alternatively, the reaction of dihydroartemisinin with a carboxylic acid as defined above may be conveniently carried out in the presence of a halogenated hydrocarbon solvent, particularly a chlorinated hydrocarbon, such as dichloromethane. The reaction may also be carried out in the presence of dicyclohexylcarbodiimide under an inert atmosphere such as a nitrogen atmosphere. Preferably, the reaction is carried out at a temperature of xe2x88x925 to +5xc2x0 C., particularly about 0xc2x0 C.
The acid chlorides, carboxylic acids and acid anhydrides of formula R5xe2x80x94COxe2x80x94W, where R5 and W are as defined above, are all known compounds or can be prepared by processes analogous to known processes.
Compounds of general formula I in which X represents a hydrogen atom, Y represents a group xe2x80x94OR6, where R6 is as defined above, and Z represents an oxygen atom may be prepared by reacting dihydroartemisinin with a compound of the general formula R4xe2x80x94OH where R6 is as defined above.
The reaction of dihyaroartemisinin with an alcohol of formula R6xe2x80x94OH as defined above may be conveniently carried out in the presence of a solvent. Suitable solvents include ethers, especially aliphatic ethers, such as diethyl ether. The reaction may also be carried out in the presence of a Lewis acid, such as boron trifluoride diethyl etherate, under an inert atmosphere, for instance, under nitrogen. Preferably, the reaction is carried out at room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
The reaction of dihydroartemisinin with an alcohol of formula R-OH as defined above may also be conveniently carried out in the presence of a cyclic ether solvent such as tetranydroturan. In this case, the reaction may also be carried out in the presence of triphenylphosphine and diethyl azodicarboxylate under an inert atmosphere, for instance, under nitrogen. Preferably, the reaction is carried out at a temperature of xe2x88x925xc2x0 C. to room temperature, preferably 0xc2x0 C. to room temperature. Ideally, the reagents are initially mixed together at 0xc2x0 C. and the reaction mixture is then allowed to warm to room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 35xc2x0 C.
In some circumstances, it may be necessary to protect certain groups of the dihydroartemisinin and/or alcohol starting materials with a protecting group to reduce the likelihood of undesirable side reactions. Suitable protecting groups include trimethylsilyl groups. In such cases, the reaction is preferably carried out in a halogenated hydrocarbon solvent, particularly a chlorinated hydrocarbon, such as dichloromethane. Preferably, the reaction is carried out in the presence of a catalytic amount of trimethylsilyl trifluoromethanesulphonate (TMSOTf) solution under an inert atmosphere, such as a nitrogen atmosphere. Preferably, the reaction is carried out at a temperature of xe2x88x9270 to 80xc2x0 C., especially about xe2x88x9278xc2x0 C. Alternatively, this reaction may be carried out at a temperature of xe2x88x925 to +5xc2x0 C, especially about 0xc2x0 C., if it is desired to increase the quantity of one of the isomers, usually the xcex2-anomer, and to reduce the quantity of the side products obtained. The trimethylsilyl protected starting materials may be prepared by reacting the dihydroartemisinin and/or alcohol with a halotrimethylsilane, such as chlorotrimethylsilane, in the presence of a base, such as pyridine or triethylamine. Preferably, the reaction is carried out at room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
Alternatively, if it is desired to increase the quantity of the other isomer, usually the xcex1-anomer, the reaction of dihydroartemisinin with an alcohol of formula R6xe2x80x94OH as defined above may be conveniently carried out in the presence of a halogenated hydrocarbon solvent, particularly a chlorinated hydrocarbon, such as dichloromethane, in the absence of any protecting groups. The reaction is preferably carried out under an inert atmosphere, such as a nitrogen atmosphere. Preferably, the reaction is carried out at a temperature of xe2x88x9230 to xe2x88x9210xc2x0 C., particularly xe2x88x9225 to xe2x88x9215xc2x0 C.
The alcohols of formula R6xe2x80x94OH, where R6 is as defined above, are all known compounds or can be prepared by processes analogous to known processes.
Compounds of general formula I in which X represents a hydrogen atom, Y represents an oxo group and Z represents a group +50 NRxe2x80x2, where R7 is as defined above, may be prepared by reacting artemisinin with a compound of the general formula R7NH2 where R7 is as defined above.
The reaction may be conveniently carried out in the presence of a solvent. Suitable solvents include lower alcohols, especially a C1-C6, alcohol, such as methanol, and halogenated hydrocarbons, especially a chlorinated hydrocarbon such as dichloromethane. The reaction is preferably carried out under an inert atmosphere, such as a nitrogen atmosphere. Preferably, the reaction is carried out at a temperature of xe2x88x925 to +5xc2x0 C., especially about 0xc2x0 C. It may also be advantageous to add p-toluene sulphonic acid during the course of the reaction to ensure that any intermediate products are converted into the final cyclised lactam of formula I. Also, in some cases, it may be necessary to utilise an amine of formula R7NH2 which has been freshly distilled before the reaction in order to ensure that reaction occurs.
Alternatively, certain compounds of general formula I in which R7 represents, a group xe2x80x94CH2H2R10, where R10 represents an electron-withdrawing group, may be prepared in a Michael addition reaction by reacting 11-azaartemisinin with a compound of the general formula CH2+50 CHxe2x80x94R10, where R10 is as defined above. Preferably, the electron-withdrawing group is a nitro, cyano, formyl, alkanoyl, alkoxycarbonyl, alkylsulphinyl, alkylsulphinyl, alkylsulphonato, arylsulphinyl, arylsulphonyl or arylsulphonato group. It is also preferred that the reaction is carried out in the presence of a base, such as sodium hydroxide, and in the presence of a solvent, such as tetrahydrofuran.
The 11-azaartemisinin starting material may be prepared by reacting artemisinin with ammonia using the first process of the invention described above.
Compounds of general formula I in which X represents a group xe2x80x94NR1R2, xe2x80x94CHR8R9 or Ar, where R1, R3 R8, R9 and Ar are as defined above, Y represents an oxo group and Z represents an oxygen atom may be prepared by reacting artemisitene of the formula III 
with a compound of the general formula MNu where M is a hydrogen or an alkali metal atom or a group xe2x80x94-LHal, where L is an alkaline earth metal atom and Hal is a halogen atom, and Nu is a nucleophilic group of formula xe2x80x94NR1R2, xe2x80x94CHR8xe2x80x2R9xe2x80x2 or Ar where R1, R2 and Ar are as defined above, R8xe2x80x2 represents a hydrogen atom or an optionally substituted alkoxycarbonyl group and R9xe2x80x2 represents a nitro group or an optionally substituted alkanoyl, aroyl, alkoxycarbonyl or aryloxycarbonyl group. The alkali metal atom may be a lithium, sodium or potassium atom. However, it is particularly preferred that the alkali metal atom is a sodium or lithium, especially a lithium, atom. The alkaline earth metal atom L is preferably magnesium and the halogen atom Hal is preferably a chlorine, bromine or iodine atom.
It is especially preferred that compounds in which X represents a group xe2x80x94NR1R2 are prepared by reacting artemisitene with a metallated secondary amine of formula MNu where M is a lithium atom or a group xe2x80x94MgHal where Hal is as defined above and Nu is a nucleophilic group of formula xe2x80x94NR1R2 as defined above This reaction may be conveniently carried out in the presence of a suitable solvent. Suitable solvents include ethers, especially cyclic ethers, such a tetrahydrofuran. Preferably, the reaction is carried out at a temperature of xe2x88x9280 to xe2x88x9260xc2x0 C., particularly xe2x88x9280 to xe2x88x9270xc2x0 C., and especially xe2x88x9278xc2x0 C.
The metallated secondary amine may be conveniently prepared by reacting a secondary amine of formula HNR1R2, where R1 and R2 are as first defined above, with a suitable lithiating agent or Grignard reagent. Suitable lithiating agents include n-, sec- or tert-butyllithium or a similar alkyllithium reagent. Suitable Grignard reagents include methyl or ethyl magnesium bromide or iodide. Preferably, the reaction is carried out in the presence of a suitable solvent, particularly an ether solvent such as tetrahydrofuran or diethyl ether, at a temperature of 0xc2x0 C. or below, usually about xe2x88x9278xc2x0 C.
Compounds of the general formula I in which Z represents an oxygen atom, Y represents an oxo group and X represents a group xe2x80x94CHR8xe2x80x2R9xe2x80x2 where R8xe2x80x2 and R9xe2x80x2 are as defined above, may be conveniently prepared by reacting artemisitene with a lithiated acetyl reagent of formula MNu where M is a lithium atom and Nu is a nucleophilic group of formula xe2x80x94CHR8xe2x80x2R9xe2x80x2 as defined above. This reaction may be conveniently carried out in the presence of a suitable solvent. Suitable solvents include ethers, especially cyclic ethers, such as tetrahydrofuran. Preferably, the reaction is carried out at a temperature of xe2x88x9280 to xe2x88x9260xc2x0 C., particularly xe2x88x9280 to xe2x88x9270xc2x0 C., and especially xe2x88x9278xc2x0 C.
The lithiated acetyl reagent may be conveniently prepared by reacting an acetyl compound of formula CH2R8xe2x80x2R9xe2x80x2, where R8xe2x80x2 and R9xe2x80x2 are as defined above, with a suitable lithiating agent. Suitable lithiating agents for this reaction include lithium diisopropylamide or a similar lithium dialkylamide base or lithium hexamethyldisilazide. Preferably, the reaction is carried out in the presence of a suitable solvent, particularly an ether solvent such as tetrahydrofuran or diethyl ether, at a temperature of 0xc2x0 C. or below, usually about xe2x88x9278xc2x0 C.
Alternatively, compounds of the general formula I in which Y represents an oxo group and X represents a group xe2x80x94CHR8xe2x80x2R9xe2x80x2 where R9xe2x80x2  and R9xe2x80x2 are as defined above, may be conveniently prepared by reacting artemisitene with a reagent of formula MNu where M is a sodium atom and Nu is a nucleophilic group of formula xe2x80x94CHR8xe2x80x2R9xe2x80x2 as defined above. This reaction may be conveniently carried out under an inert atmosphere, such as nitrogen, in the presence of a suitable solvent. Suitable solvents include ethers, especially cyclic ethers, such as tetrahydrofuran. Preferably, the reaction is carried out at a temperature of xe2x88x925 to +5xc2x0 C., especially about 0xc2x0 C.
The reagent of formula MNu where M is a sodium atom and Nu is a nucleophilic group of formula xe2x80x94CHR8xe2x80x2R9xe2x80x2 as defined above may be conveniently prepared by reacting, for instance, sodium hydride with a compound of formula CH2R8xe2x80x2R9xe2x80x2, where R8xe2x80x2 and R9xe2x80x2 are as defined above.
Compounds of the general formula I in which Z represents an oxygen atom, Y represents an oxo group and X represents a group xe2x80x94CHR8xe2x80x2R9xe2x80x2 where R8xe2x80x2 and R9xe2x80x2 are as defined above, may also be conveniently prepared by reacting artemisitene with a reagent of formula MNu where M is a hydrogen atom and Nu is a nucleophilic group of formula xe2x80x94CHR8xe2x80x2R9xe2x80x2 as defined above. This reaction may be conveniently carried out in the presence of a suitable solvent. Suitable solvents include ethers, especially cyclic ethers, such as tetrahydrofuran. Preferably, the reaction is carried out in the presence of tris(dimethylamino)sulphur (trimethylsilyl)difluoride (TASF) and it is preferred that this stage of the reaction is carried out at a temperature of 0xc2x0 C. or below, usually about xe2x88x9278xc2x0 C. It may also be advantageous, or in some cases necessary, subsequently to add glacial acetic acid to the reaction mixture. This stage of the reaction, if included, may be carried out at room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
In some cases, it may be advantageous or necessary to use a silylated form of the reagent of formula MNu as defined above either to protect a functional group of the reagent during reaction or to stabilise the intermediate carbanion formed during the reaction. Suitable silylated forms of the reagent of formula MNu where M is as defined-above and Nu is a nucleophilic group of formula xe2x80x94CHR8xe2x80x2R9xe2x80x2 as defined above include compounds in which Rxe2x80x2 and R9xe2x80x2 or a portion thereof has been replaced by a trimethylsilyl group. Such compounds can be prepared by reacting a suitable compound of formula MNu as defined above with a halotrimethylsilane, such as chlorotrimethylsilane or bromotrimethylsilane.
Compounds of the general formula I in which W represents an oxo group and X represents a group Ar, where Ar is as defined above, maybe conveniently prepared by reacting artemisitene with a reagent of formula QNu where Q is an alkali metal atom, preferably lithium, or a group xe2x80x94MHal where M is an alkaline earth metal atom, preferably magnesium, and Hal is a halogen atom. This reaction may be conveniently carried out in the presence of a catalytic amount of a copper (I) salt, such as copper (I) iodide, in a suitable solvent. Suitable solvents include ethers, such as diethyl ether and, especially, cyclic ethers, such as tetrahydrofuran. Preferably, the reaction is carried out at a temperature at or below 0xc2x0 C., preferably about xe2x88x9210xc2x0 C. In this reaction, the metallated Ar group adds to the exocyclic double bond of artemisitene to form an enolate, which is converted into the desired product of formula I when the reaction mixture is treated with a proton source, such as aqueous ammonium chloride.
The aryl Grignard or aryllithium reagent of formula QNu as defined above may be conveniently prepared by treating a compound of general formula ArHal, where Ar and Hal are as defined above, with either magnesium or lithium metal. Preferably, the reaction is carried out in the presence of a suitable solvent, preferably an ether solvent, such as diethyl ether, tetrahydrofuran or dimethoxyethane.
Suitable reducing agents for forming compounds of the general formula I in which Zxe2x80x2 represents an oxygen atom, Y represents a hydrogen atom and X represents a group where R8xe2x80x2 and R9xe2x80x2 are as defined above, include sodium borchydride in the presence of boron trifluoride diethyl etherate, diisobutylaluminium hydride, similar Lewis acidic metal hydrides and triethylsilane. The reduction reaction may be conveniently carried out in the presence of a suitable solvent. Suitable solvents include ethers, especially cyclic ethers, such as tetrahydrofuran. Preferably, the reaction is carried out at a temperature of xe2x88x925xc2x0 C. to the reflux temperature of the reaction mixture, especially 0xc2x0 C. to reflux temperature. Ideally, the reagents are initially mixed together at 0xc2x0 C. and the reaction mixture is then subsequently heated at reflux temperature.
Depending on the reducing agent and reaction conditions selected, the carbonyl group in the R9xe2x80x2 moiety of compounds of the general formula I in which Z represents an oxygen atom, Y represents an oxo group and X represents a group xe2x80x94CHR8xe2x80x2R9xe2x80x2, where R8xe2x80x2 and R9xe2x80x2 are as defined above, may also be reduced to give compounds of the general formula I in which Z represents an oxygen atom, Y represents a hydrogen atom and X represents a group xe2x80x94CHR8xe2x80x2R9xe2x80x2 where R8xe2x80x2 is as defined above and R9xe2x80x2 is a group xe2x80x94CH2RE where RE is an alkyl, aryl, alkoxy or aryloxy group.
Artemisitene may be prepared by reacting 10-hydroperoxy-10-dihydroartemisitene (9-hydroperoxyartemisitene) of formula 
with acetic anhydride in the presence of a base, preferably pyridine. Preferably, the reaction is carried out at room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
10-Hydroperoxy-10-dihydroartemisitene (9-hydroperoxyartemisitene) may be prepared by reacting 9,10-arhydro-10-deoxoartemisinin (9,10-anhydrodehydroartemisinin) of formula 
with oxygen in the presence of a solvent, preferably a halogenated hydrocarbon, such as dichloromethane: A photosensitiser, such as methylene blue, is preferably included in the reaction mixture to convert groud state (triplet) oxygen into excited state (singlet) oxygen under irradiation from a light source. The active agent which converts the 9,10-anhydro-10-deoxoartemisinin (9,10-anhydrodehydroartemieninin) into 10-hydroperoxy-10-dihydroartemisitene (9-hydroperoxyartemisitene) is therefore singlet oxygen.
9, 10-Anhydro-10-deoxoartemisinin(9,10-anhy(irodehydroartemisinin) may be prepared by reacting dihydroartemisinin with a dehydrating agent, such as boron trifluoride diethyl etherate, preferably in the presence of a solvent, especially an ether solvent, such as diethyl ether. Ideally, the reaction is, carried out at room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
9,10-Anhydro-10-deoxoartemisinin (9,10-anhydroartemisinin) may also be conveniently prepared by reacting dihydroartemisinin with trifluoroacetic anhydride. The reaction may be conveniently carried out in the presence of a solvent, preferably a halogenated hydrocarbon, and especially a chlorinated hydrocarbon, such as dichloromethane. It is also preferred that the reaction is carried out in the presence of a base, such as pyridine or a derivative thereof, for example, dimethylamino-pyridine. Preferably, the reaction is carried out under an inert atmosphere, such as nitrogen, at a temperature of xe2x88x925xc2x0 C. to +5xc2x0 C., preferably 0xc2x0 C., with the reaction mixture being subsequently allowed to warm to room temperature, that is, 15 to 35xc2x0 C., preferably 20 to 30xc2x0 C.
The amines of formula HNR1R2, acetyl compounds of formula CH2R8xe2x80x2R9xe2x80x2 and aryl halide compounds of formula ArHal, where R1, R2, R8xe2x80x2, R9xe2x80x2 and Ar are as defined above, are all known compounds or can be prepared by processes analogous to known processes.
Compounds of the general formula I in which X represents a group xe2x80x94CHR8R9, where R8 and R9 are as first defined above, can also be prepared by reacting artemisitene with a compound of the general formula TCHR8R9, where T represents a halogen atom and R8 and R9 are as defined above, to form a compound of formula I in which Y represents an oxo group and X is as defined above; and, if desired, reacting the compound of formula I thus formed with a suitable reducing agent to form a compound of formula I in which Y represents a hydrogen atom and X is as defined above.
Preferred compounds of formula TCHR8R9 are those in which T represents a chlorine or bromine, especially a bromine, atom. It is also preferred that this reaction is carried out in the presence of a suitable solvent. Suitable solvents include ethers, such as 1,2-dimethoxyethane. Preferably, the reaction is carried out in the presence of catalytic amounts of an initiator, such as 2,2xe2x80x2-azobisisobutyronitrile (AIBN, also known as 2,2xe2x80x2-azobis[2-methylpropane-nitrile]), in the presence of tri-n-butyltin hydride. In this context, AIBN converts tri-n-butyltin hydride into the tributyltin radical which abstracts halogen from the compound of formula TCHR8R9 to provide a carbon radical which adds to the exocyclic double bond of artemisitene. After addition, the resulting artemisinin radical is reduced by hydrogen atom transfer from the tri-n-butyltin hydride and the chain process is maintained by the tributyltin radical. It is also preferred that the reaction is carried out at a temperature of 60 to 100xc2x0 C., particularly 70 to 90xc2x0 C., and preferably 75 to 85xc2x0 C.
Suitable reducing agents for forming compounds of the general formula I in which Y represents a hydrogen atom and X represents a group xe2x80x94CHR8R9, where R8 and R9 are as defined above, include sodium borohydride in the presence of boron trifluoride diethyl etherate, diisobutylaluminium hydride, similar Lewis acidic metal hydrides and triethylsilane. The reduction reaction may be conveniently carried out in the presence of a suitable solvent, suitable solvents including ethers, such as tetrahydroturan. Preferably, the reaction is carried out at a temperature of xe2x88x925xc2x0 C. to the reflux temperature of the reaction mixture, especially 0xc2x0 C. to reflux temperature. Ideally, the reagents are initially mixed together at 0xc2x0 C. and the reaction mixture is then subsequently heated at reflux temperature.
Artemisitene can be prepared as described above and compounds of formula TCHR8R9 are known compounds or can be prepared by processes analogous to known processes.
The invention also provides a pharmaceutical composition which comprises a carrier and, as active ingredient, a novel compound of the general formula I as defined above.
A pharmaceutically acceptable carrier may be any material with which the active ingredient is formulated to facilitate administration. A carrier may be a solid or a liquid, including a material which is normally gaseous but which has been compressed to form a liquid, and any of the carriers-normally used in formulating pharmaceutical compositions may be used. Preferably, compositions according to the invention contain 0.5 to 95% by weight of active ingredient.
The compounds of general formula I can be formulated as, for example, tablets, capsules, suppositories or solutions. These formulations can be produced by known methods using conventional solid carriers such as, for example, lactose, starch or talcum or liquid carriers such as, for example, water, fatty oils or liquid paraffins. Other carriers which may be used include materials derived from animal or vegetable proteins, such as the gelatins, dextrins and soy, wheat and psyllium seed proteins; gums such as acacia, guar, agar, and xanthan; polysaccharides; alginates; carboxymethylcelluloses; carrageenans; dextrins; pectins; synthetic polymers such as polyvinylpyrrolidone; polypeptide/protein or polysaccharide complexes such as gelatin-acacia complexes; sugars such as mannitol, dextrose, galactose and trehalose; cyclic sugars such as cyclodextrin; inorganic salts such as sodium phosphate, sodium chloride and aluminium silicates; and amino acids having from 2 to 12 carbon atoms such as a glycine, L-alanine, L-Aspartic acid, L-glutamic acid, L-hydroxyproline, L-isoleucine, L-leucine and L-phenylalanine.
Auxiliary components such as tablet disintegrants, solubilisers, preservatives, antioxidants, surfactants, viscosity enhancers, colouring agents, flavouring agents, pH modifiers, sweeteners or taste-masking agents may also be incorporated into the composition. Suitable colouring agents include red, black and yellow iron oxides and FD and C dyes such as FD and C blue No. 2 and FD and C red No. 40 available from Ellis and Everard. Suitable flavouring agents include mint, rasberry, liquorice, orange, lemon, grapefruit, caramel, vanilla, cherry and grape flavours and combinations of these. Suitable pH modifiers include citric acid, tartaric acid, phosphoric acid, hydrochloric acid and maleic acid. Suitable sweeteners include aspartame, acesulfame K and thaumatin. Suitable taste-masking agents include sodium bicarbonate, ion-exchange resins, cyclodextrin inclusion compounds, adsorbates or microencapsulated actives.
According to another aspect of the invention there is provided a compound of the general formula Axe2x80x94B in which A represents a ligand which is capable of binding to a nucleic acid and B is a group containing a trioxane moiety which is capable of acting as a source of a free radical which free radical is capable of chemically interacting with a nucleic acid.
Preferably, the free radical is an oxygen-centred or a carbon-centred free radical and the trioxane moiety acts as a controlled source of such free radicals. Typically, a free radical of this type is formed by a mechanism in accordance with or similar to the mechanisms set out in Scheme 1 or Scheme 2 above, that is, reduction of the peroxide bond by access to a one-electron reductant, such as Fe(II) or an organic reductant such as a thiol, or ring opening to provide hydroperoxide and then hydroxyl radical or, perferryl iron.
It is desirable that ligand A should be capable of aligning the trioxane moiety proximate to specified base sequences in DNA. The binding of ligand A to a nucleic acid may be accomplished by a variety of methods in order to achieve this aim. Thus, it is preferred that A represents a ligand which is capable of intercalating into a nucleic acid, binding to the minor groove of a nucleic acid or bonding to a deoxyribose or phosphate group of a nucleic acid. The bonding of the ligand A to a deoxyribose or phosphate group of a nucleic acid ideally takes place by means of hydrogen bonding.
In the case of ligands which act as intercalators, it is important that the trioxane moiety be attached to the intercalating group in a manner which does not affect its intercalation properties. Thus, the trioxane moiety must not interact chemically with the intercalating group upon activation of the peroxide moiety within the trioxane pharmacophore.
Typical ligands which are capable of binding to a nucleic acid include optionally substituted aromatic, polycyclic aromatic, glycoside and polypyrrole groups.
The group containing a trioxane moiety is preferably derived from artemisinin or an analogue thereof. However, it may also be derived from a synthetic trioxane.
In one preferred group of compounds, the compound of general formula Axe2x80x94B is a compound of the general formula IV 
or a salt thereof, in which A is as defined above.
Preferably, A represents an optionally substituted aryl or C-linked heteroaryl group or a group xe2x80x94NR3R4, xe2x80x94Oxe2x80x94COxe2x80x94R5 or xe2x80x94OR6; where R3 represents a hydrogen atom or an optionally substituted alkyl group and R4 represents an optionally substituted aryl or aralkyl group, or R3 and R4 together with the interjacent nitrogen atom represent an optionally substituted heterocyclic group; R5 represents an optionally substituted aryl, aralkyl, heterocyclic or polycyclic group; and R6 represents an optionally substituted aryl, aralkyl, heterocyclic or polycyclic group.
In one preferred aspect, A represents a C6-18 aryl group or a 5- to 10-membered C-linked heteroaryl group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-4 alkyl, C2-4 alkenyl, C1-4 haloalkyl, C1-4 alkoxy, C1-4 haloalkoxy, amino, C1-4 alkylamino, di(C1-4alkyl)amino and carboxyl groups.
In a particularly preferred sub-group of these compounds, A represents a phenyl, chlorophenyl, bromophenyl, trimethylphenyl, vinylphenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl, carboxylphenyl, naphthyl, hydroxynaphthyl, anthryl or phenanthryl group. Compounds in which A represents a dimethoxyphenyl or trimethoxyphenyl group are especially preferred.
In another preferred aspect, A represents a group xe2x80x94NR3R4 where R3 represents a hydrogen atom or a C1-6 alkyl group and R4 represents a C6-10 aryl or C7-16 aralkyl group, or R3 and R4 together with the interjacent nitrogen atom represent a 5- to 10-membered heterocyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C1-4 haloalkyl, C1-6 alkoxycarbonyl and phenyl groups.
In a particularly preferred sub-group of these compounds, A represents a phenylamino, fluorophenylamino, chlorophenylamino, bromophenylamino, iodophenylamino, methoxycarbonylphenylamino, biphenylamino, benzylamino, fluorobenzylamino, bis(trifluoromethyl)benzylamino, morpholinyl, thiomorpholinyl, morpholinosulphonyl, indolinyl or tetrahydroisoquinolinyl group. Compounds in which A represents a phenylamino or fluorophenylamino group are especially preferred.
In a further preferred aspect, A represents a group xe2x80x94Oxe2x80x94COxe2x80x94R5 where R5 represents a C6-18 aryl, 5- to 18-membered heterocyclic or C4-26 polycyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy, C1-4 haloalkoxy, oxo, carboxyl and carboxylato groups. More preferably, A represents a group xe2x80x94Oxe2x80x94COxe2x80x94R5 where R5 represents a C6-14 aryl, 5- to 14-membered heterocyclic or C6-14 polycyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-4 alkyl, C1-4 haloalkyl, oxo and carboxylato groups.
In a particularly preferred sub group of these compounds, A represents a group xe2x80x94Oxe2x80x94COxe2x80x94R5 where R5 represents a phenyl, hydroxynaphthyl, anthryl, anthraquinonyl, quinolinyl, isoquinolinyl, quinoxalinyl or acridinyl group.
In another preferred aspect, A represents a group xe2x80x94OR6 where R6 represents a C6-24 aryl, C7-30 aralkyl, 5- to 18-membered heterocyclic or C4-26 polycyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-8 alkyl, C1-4 haloalkyl, C1-4 alkoxy, C1-4 haloalkoxy, carboxyl and C1-4 alkoxycarbonyl groups. More preferably, A represents a group xe2x80x94OR6 where R6 represents a C6-14 aryl, C7-18 aralkyl, 5- to 14-membered heterocyclic or C6-18 polycyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy and C1-4 haloalkoxy groups.
In a particularly preferred sub-group of these compounds, A represents a group xe2x80x94OR6 where R6 represents a phenyl, methoxyphenyl, naphthyl, benzyl, fluorobenzyl, naphthylmethyl, anthrylmethyl, phenanthrylmethyl, pyrylmethyl, quinolinyl, trifluoromethylquinolinyl or cholestenyl group. Compounds in which A represents a group xe2x80x94OR6 where R6 represents a naphthylmethyl group are especially preferred.
In another preferred group of compounds, the compound of general formula A-B is a compound of the general formula V 
in which A is a ligand which is capable of binding to a nucleic acid.
Preferably, A represents a C7-16 aralkyl or 5- to 10-membered-heterocyclic-C1-6 alkyl group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, cyano, nitro, C1-4 haloalkyl, formyl, C1-6 alkoxycarbonyl, C1-6 alkanoyl, phenylsulphinyl, phenylsulphonyl and phenylsulphonato groups. More preferably, A represents a benzyl group optionally substituted by one or more halogen atoms or hydroxyl groups.
In a particularly preferred sub-group of these compounds, A represents a benzyl or fluorobenzyl group.
In a further preferred group of compounds, the compound of general formula A-B is a compound of the general formula VI 
in which A is a ligand which is capable of binding to a nucleic acid.
It is preferred that A represents a group xe2x80x94CH2xe2x80x94NR1R2 where one of R1 and R2 represents an optionally substituted aryl or aralkyl group and the other of R1 and R2 represents an optionally substituted alkyl, cycloalkyl, aryl or aralkyl group, or R1 and R2 together with the interjacent nitrogen atom represent an optionally substituted heterocyclic group or an amino group derived from an optionally substituted amiono acid ester which contains an aromatic or heterocyclic moiety. Suitable amino acids in this respect include histidine, phenylalanine, tyrosine, tryptophan, proline and hydroxyproline.
Preferably, A represents a group xe2x80x94CH2xe2x80x94NR1R2 where one of R1 and R2 represents a C6-10 aryl or C7-16 aralkyl group and the other of R1 and R2 represents a C1-6 alkyl, C6-10 aryl or C7-16 aralkyl group, or R1 or R2 together with the interjacent nitrogen atom represent a 3- to 14-membered heterocyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C1-4 haloalkyl and C1-6 alkoxycarbonyl groups. More preferably, A represents a group xe2x80x94CH2xe2x80x94NR1R2 where one or R1 and R2 represents a C7-10 aralkyl group and the other of R1 and R4 represents a C1-4 alkyl or C7-10 aralkyl group,, or R1 and R4 together with the interjacent nitrogen atom represent a 5- to 10-membered heterocyclic group, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, C1-4 alkyl, C1-4 haloalkyl and C1-4 alkoxycarbonyl groups.
In a particularly preferred sub-group of these compounds, A represents a dibenzylaminomethyl or indolinylmethyl group.
In formula VI, A may also represent a group xe2x80x94CH2xe2x80x94Ar where Ar is as defined above.
In another preferred group of compounds, the compound of general formula A-B is a compound of the general formula VII 
in which A is a ligand which is capable of binding to a nucleic acid and Y is a hydrogen atom or an oxo or hydroxyl group.
Preferably, A represents a group xe2x80x94CH2xe2x80x94CR8R9 where R8 represents a hydrogen atom or an optionally substituted alkyl, alkenyl, alkynyl, aryl or alkoxycarbonyl group; and R9 represents a nitro group or an optionally substituted alkyl, alkenyl, alkynyl, aryl, alkanoyl, aroyl, alkoxycarbonyl or aryloxycarbonyl group, with the proviso that at least one or R8 and R9 represents or contains an aromatic moiety, or R8 and R9 together with the interjacent carbon atom represent an optionally substituted polycyclic group containing an aromatic moiety; with the proviso that, when R8 represents a hydrogen atom and R9 represents a benzyl or 4-chlorobenzyl group, then Y represents a hydroxyl group. More preferably, A represents a group xe2x80x94CH2xe2x80x94CR8R9 where R8 represents a hydrogen atom or a C1-6 alkyl or C1-6 alkoxycarbonyl group and R9 represents a C7-11 aroyl or C6-10 aryloxycarbonyl group, or R8 and R9 together with the interjacent carbon atom represent a C4-26 polycyclic group containing an aromatic moiety, each group being optionally substituted by one or more substituents selected from the group consisting of halogen atoms, hydroxyl, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy and C1-4 haloalkoxy groups.
In formula VII, A may also represent a group xe2x80x94CH2xe2x80x94Ar where Ar is as defined above.
It should also be appreciated that many of the compounds of the general formula A-B are capable of existing as different geometric and optical isomers. The present invention thus includes both the individual isomers and mixtures of such isomers.
Compounds of general formula A-B as defined above may be prepared by the processes described earlier in this specification or by processes analogous to such processes.
At present, drug research relies to a large extent on ad hoc processes involving the uncovering of new chemical entities from nature and post-hoc establishment of mode of action, the ene-diyne antibiotics discussed earlier being good examples in this respect. Often a single drug candidate compound will be isolated from a natural source and structural variation must be carried out to modify undesirable properties. However, it is an advantage of the present invention that families of compounds can be easily prepared which are based on the trioxane pharmacophore and the biological activity of such compounds can be varied through variation in the nature of the binding group attached to the trioxane moiety thereby enabling different drug targets to be selected. Considerable structural variation in target compounds is possible through relatively straightforward structural variation in the ligand and trioxane moiety and this allows for fine tuning of biological activity. Moreover, since the starting materials are commercially available and the chemical transformations required to transform them into drug candidate compounds are relatively straightforward, the compounds are relatively accessible.
A further advantage of the compounds of the invention is that the compounds themselves provide a source of active free radicals and do not therefore require activation as in the case of other drugs, such as bleomycin. The utility of new drugs containing such a radical generating xe2x80x9cwarheadxe2x80x9d is potentially enormous, particularly in the targeting of tumours. Moreover, since the compounds do not require activation, they have potential use against hypoxic cells, such as in solid tumours. It has also been shown that the compounds are more cytotoxic against rapidly replicating cells making them particularly attractive for use as antitumour agents. Moreover, many of the compounds have been found to damage DNA, particularly intracellular DNA.
The invention also provides a pharmaceutical composition which comprises a carrier and, as active ingredient, a compound of the general formula A-B as defined above. Such compositions may contain the same ingredients and be formulated in the same way as the compounds of general formula I described above.
Many of the compounds of formula A-B described above, especially those falling within the particularly preferred sub-groups of compounds, have been found to exhibit cytotoxic activity. Such compounds appear to act by selective destruction of DNA in tumour cells. The invention therefore further provides a compound of the general formula A-B as defined above for use as a cytotoxic agent, for use as an antitumour agent and/or for use in the treatment of cancer. The invention also provides the use of a compound of the general formula A-B as defined above for the manufacture of a medicament for use as a cytotoxic agent, for use as an antitumour agent and/or for the treatment of cancer.
The invention also provides a method for killing a cell which comprises exposing the cell to a compound of the general formula I or a compound of the general formula A-B as defined above. A method for treating cancer is also provided which comprises administering to an animal in need of such treatment a therapeutically effective amount of a compound of the general formula I or a compound of the general formula A-B as defined above. Preferably, the animal is a mammal, especially a human.
The invention is further illustrated by the following examples.